Assay device

ABSTRACT

An assay device and method are provided which allow the determination of the presence or absence of at least one analyte in a test sample, while providing specific identification of the test subject. The assay device includes a reaction medium having at least one reaction zone and at least one control zone, which is capable of providing a pattern suitable for identifying the test subject. The pattern suitable for identifying the test subject is preferably a fingerprint. In a preferred embodiment of the invention, the reaction zone and the control zone include at least one member of a ligand/receptor pair.

This application is a continuation of U.S. Ser. No. 10/061,351, filedFeb. 4, 2002, now abandoned; which is a divisional of U.S. Ser. No.08/788,343, filed Jan. 27, 1997, now U.S. Pat. No. 6,352,863; which is acontinuation of U.S. Ser. No. 08/484,845, filed Jun. 7, 1995, nowabandoned; which is a continuation of U.S. Ser. No. 08/123,077, filedSep. 17, 1993, now abandoned; which is a continuation-in-part of U.S.Ser. No. 08/118,268 filed Sep. 9, 1993, now abandoned; which is acontinuation of U.S. Ser. No. 07/759,922, filed Sep. 13, 1991, now U.S.Pat. No. 5,244,815; which is a continuation-in-part of U.S. Ser. No.07/668,115, filed Mar. 12, 1991, now abandoned; which is acontinuation-inpart of U.S. Ser. No. 07/467,532, filed Jan. 19, 1990,now abandoned.

TECHNICAL FIELD OF THE INVENTION

The invention is in the field of ligand-receptor assays, includingimmunoassays, used for determining the presence or absence of an analytein a biological fluid. More particularly, the apparatuses and methods ofthe invention relate to establishing the identity of a test subject anddetermining the presence or absence of at least one analyte in abiological fluid taken from the test subject using a single test device.

BACKGROUND OF INVENTION

For many years, those skilled in the art of ligand-receptor assays havesought an effective, inexpensive, and reliable device and method fordetecting the presence and/or absence of antigens, antibodies, and thelike. The art is replete with such devices.

For many years, those skilled in the art have also sought devices andmethods for assuring that the sample being tested actually came from acertain individual. Many schemes, some elaborate, some simple, have beenestablished to verify that the test sample was in fact produced by aparticular individual. Similarly, many schemes exist to thwart matchinga particular test result with a particular person.

For example, drug testing is now a routine procedure in athletics,prison, and the work place. The protocols involve testing individualfluid samples such as urine or blood to determine the presence ofcertain antibodies in the fluid sample; a positive result may be anindication of drug use.

Previously such testing has been accomplished by a series of tests whichmay involve shifting of the fluid being tested to different containersand removal of the fluid from the person being tested to a distantplace. Oftentimes, fluid misplaccement and/or substitution openedquestions as to the chain of custody of the tested fluid. Thus, aproblem which has occurred during such testing is that test fluids maybe obtained from persons other than the person to be tested or that testfluids become mixed, lost, or cannot be specifically identified withthat person after the test is returned from the laboratory. Also, theseassays typically take too long to obtain timely results.

Other shortcomings are associated with many of the devices and methodscurrently used for the determination of analytes in biological fluids.Frequently, a certain level of skill is required to use or handle suchdevices to obtain satisfactory results. In addition, most of thesedevices require one or more additional reagents, and frequently a washsolution, to apply to the device during the course of the test. In manyof these devices, due at least in part to the limited sensitivity of thedevices, relatively large amounts of oftentimes expensive reagents or oftest sample are required to give accurate results.

As noted above, the principles which form the basis for this type ofdetection is a person's immune system, i.e., the inherent capability ofa mammal to respond to a foreign molecule, typically a macromolecule.Hereinafter, any molecule which is capable of eliciting such a responsewill be referred to as an antigen, i.e., an antibody generator. Theproteins and protein fragments which are produced in response to theantigen will be referred to as antibodies or immunoglobulins.

SUMMARY OF THE INVENTION

The present invention overcomes problems inherent in known assay devicesand systems through the use of a specifically designed assay device,preferably an immunoassay device which allows the determination of thepresence or absence of an analyte in a sample, and preferablyspecifically identifies the person providing the sample.

The present invention is directed to a testing method and device andmore specifically to a method and device for detecting the presence ofan analyte, most preferably specific antigens or specific antibodies ina biological fluid. The present invention preferably also provides forpositively identifying the individual tested.

The present invention provides an easily handled, disposable testing padand a disposable test device suitable for detecting the presence orabsence of an analyte and preferably for identifying the test subject.For example, a finger of the test subject may be coated with a labelledligand and the finger may be pressed on a membrane substrate having aspecific immobilized ligand bed to capture an analyte from a biologicalfluid while simultaneously providing an inkless (specific binding)visible fingerprint of the test subject so that positive identificationof the fluid donor is irrefutably obtained. The fingerprint of the fluiddonor obtained on the membrane may also include information aboutanalytes, such as drugs, present in his body fluids at the time ofperforming the test. This information can be recorded or stored forpositive identification when needed.

It is an object of the invention to assay a test sample for the presenceor absence of an analyte while also identifying the subject beingtested. Thus, problems of misidentification, chain of title, and delaysmay be eliminated.

It is an object of the present invention to provide a device for rapidlydetermining the presence or absence of an analyte taken from a testsubject. It is also an object of the present invention to provide adevice for rapidly determining the quantitative amount of an analyte ina sample taken from a test subject. Yet other objects of the presentinvention are to provide a device and a method of determining thepresence or absence of a drug or drug metabolite in a test sample takenfrom a test subject and to provide a device and method determiningwhether a test subject is an acute or chronic drug abuser.

Another embodiment of the present invention, where identifying the testsubject by a fingerprint is not critical, involves applying the sampleof body fluid or a labelled ligand to the membrane with the pressure ofa transfer means or applicator that is part of the test device. Incontrast to similar known devices which apply liquid test samples and/orreagents dropwise to the surface of a reaction medium, the presentinvention, in which liquid test samples and/or reagents are applied andmaintained under pressure on the surface of the reaction medium for ashort duration, requires smaller amounts of test sample and reagents anddemonstrates higher sensitivities than known devices.

A device according to the present invention which uses this principle isan enclosed assay test device. A preferred embodiment is one in whichthe assay test device includes a housing, a reaction medium having atleast one reaction zone located in the housing, a medium containing asignal-producing or analyte-indicating agent located in the housing, anda medium for containing a test sample located in the housing. Both themedium containing a signal-producing agent and the medium for containinga test sample located in the housing are movable independently between afirst position in spaced relationship to the reaction medium and asecond position in substance-transferrable contact with the reactionmedium. Preferably this is a self-contained test device.

A particular embodiment of this invention includes an assay test deviceemploying a two-part housing. In one embodiment of the device, areaction medium having at least one reaction zone is mounted in a firstpart of the housing. In the second part of the two-part housing islocated a medium for containing a portion of a test sample. The mediumfor containing a portion of a test sample and the reaction medium are soarranged in the two parts of the housing that when the housing parts arebrought together in a closed relationship, there is substancetransferrable contact between the medium for containing a portion of atest sample and the reaction medium. In addition to at least onereaction zone, the reaction medium preferably has a control zone whichis provided to identify the test subject by a fingerprint or like meansif desired. Such a device may be designed as a self-contained assaydevice. As used herein, the term “self-contained” means that the devicemay be used without the addition of additional reagents or solutions, oronly requiring the dropwise addition of water.

Another embodiment employs a somewhat similar arrangement but a mediumcontaining a signal-producing agent is substituted for the medium forcontaining a portion of a test sample.

A preferred device according to the above “pressure” principle issimilar to the aforementioned devices in that a two-part housing is alsoemployed in which a reaction medium having at least one reaction zone ismounted in a first part of the two-part housing. However, like one ofthese embodiments, in the second part of the two-part housing, there islocated a medium containing a signal-producing agent, such as a labelledmaterial which will indicate the presence of an analyte. The device alsoincludes a removable medium for containing a portion of a test samplewhich is located intermediate the first and second parts of the housingand in substance-transferable contact with the reaction medium when thefirst and second parts of the housing are in a closed relationship in afirst closed position of the housing. The reaction medium and mediumcontaining an analyte-indicating agent (or signal-producing agent) areso positioned and arranged within their separate housing parts that whenthe parts of the housing are in a closed relationship in a secondposition, the reaction medium and means for transferring theanalyte-indicating agent are in substance transferable contact with oneanother.

In using this device, a test sample drawn from a test subject is placedon a removable medium for containing and transferring a portion of atest sample which is placed in the two-part housing. The housing is thenclosed to a first position such that the removable test samplecontaining medium is in transferable contact with and applies pressureto the reaction medium. The housing is then opened and the removablemedium for containing and transferring a test sample is removed from thehousing. The housing is closed a second time to a second closed positionsuch that the means for transferring an analyte-indicating agent is intransferable contact with and applies pressure to the reaction medium.

In another embodiment of the invention, various segregated areas of thereaction medium include a specific member of an immunological pair (MIP)which bind to or capture a specific analyte, such as a drug or drugmetabolite. Different segregated areas may contain MIPs that arespecific for different designated analytes, thereby allowingdeterminations for more than one analyte to be carried out in a singleassay.

The methods and devices according to the invention are particularlybeneficial in that only a small amount of signal-producing agent isrequired, in contrast to the conventional flow through cassettes ordevices; applying pressure while maintaining direct contact on thesurface of the test device maintains a localized concentration ofreagents, particularly the signal-producing agent, which appears toincrease the speed and sensitivity of the reaction, e.g., test resultsare obtained in a matter of seconds versus a matter of minutes orlonger; and, in some embodiments, labeling a secondary antibody (asopposed to labeling the primary antibody or the antigen) decreases thereaction time of the assay because the antigen's shape is not altered bybeing bonded to a label.

DEFINITIONS

1. LIGAND-RECEPTOR ASSAY: any technique involving the detection of thecomplex formed between a ligand and a substance which binds to theligand. Preferably, one member of the complex is an analyte. Thepreferred ligand-receptor assay is an immunoassay. Ligand-receptorassays may be used to determine the presence, absence, quantity, andconcentration of ligands in biological fluids. Ligand-receptor assaysmay be competitive or non-competitive, homogeneous or heterogeneous,direct or indirect, or a combined detection technique, e.g., binding anantibody to a small chemical moiety such as biotin or dinitrophenol(DNP).

It is preferred that during the assay process, substantially all of atleast one predetermined ligand or ligand receptor remains in apredetermined position. Any technique for immobilizing a ligand orligand receptor is included in the scope of the present invention. In apreferred embodiment, a ligand or ligand receptor is bound orimmobilized on or in a solid phase. Typical immobilization mechanismsinclude, but are not limited to, covalent binding, noncovalent binding,chemical coupling, physical entrapment, and adsorption.

2. LIGAND: refers to the analyte itself, or a substance which can beused to infer the presence of an analyte in a test sample. Aligand-receptor refers to the substances for which there is a specificbinding partner, e.g., the ligand. As used herein, ligand andligand-receptors may be members of an immunological pair (MIP). Ligandsand ligand-receptors may include haptens, hormones, antigens, antibodies(including anti-antibodies and antibody fragments such as Fab or Fc),DNA, RNA, oligonucleotides, nucleic acids, and complexes or metabolitesof any of the above. The ligand or the ligand-receptor may be labeled orunlabeled.

3. ANALYTE: the substance to be assayed, and, depending on the specificassay used, may be a ligand or a ligand-receptor. Exemplary analytesinclude but are not limited to drugs, proteins, haptens, hormones,metabolites of the aforementioned, and other molecules, alone or incombination with a protein. A hapten is a small molecular weightmaterial, such as some drugs or drug metabolites, which typically firstattach to a protein in order to be antigenic or immunogenic. Forexample, some drugs may be found in the body in both free and boundforms, which in turn provides the ability to distinguish between chronicdrug abusers and acute drug abusers. In the case of the former, in manyinstances an antibody is formed in the body against the bound form whichcan also be detected as an analyte by the present invention.

4. BIOLOGICAL FLUID: any body fluid which may be tested to determine thepresence or absence of an analyte, including, but not limited to bloodor a blood component, saliva, urine.

5. Signal-producing AGENT: any agent used in a ligand-receptor assaywhich can be used to produce a detectable signal. The preferredsignal-producing agents provide an easily visible signal, and morepreferably a color.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of an assay device according to the presentinvention.

FIG. 2 is a plan view of an assay device according to the presentinvention, illustrating the results of conducting a test for thepresence or absence of an antigen and the formation of a patternsuitable for identifying a test subject.

FIG. 3 is an enlarged schematic cross sectional view of an assay deviceaccording to the present invention, showing a reaction zone in areaction medium.

FIG. 4 is an enlarged schematic cross sectional view of an assay deviceaccording to the present invention, showing a control zone in a reactionmedium.

FIG. 5 is an enlarged schematic cross sectional view of an assay deviceaccording to the present invention, illustrating a positive test result.

FIG. 6 is an enlarged schematic cross sectional view of an assay deviceaccording to the present invention, illustrating a negative test result.

FIG. 7 is an abstract representation of a cross section of an assaydevice according to the present invention, illustrating contacting theassay device method according to the invention.

FIG. 8 is an abstract representation of a cross section of animmunoassay device according to the present invention, illustrating theformation of a fingerprint in a control zone of the reaction medium.

FIG. 9 is an abstract representation of a cross section of an anotherembodiment of an immunoassay device according to the present invention,illustrating the transfer of a test mixture from a swab to a finger ofthe person providing the test sample.

FIG. 10 is an abstract representation of a cross section of theimmunoassay device of FIG. 9, illustrating the application of the testmixture to the reaction medium by the finger of the person providing thetest sample.

FIG. 11 is an open, exploded perspective view of one embodiment of adevice according to the present invention.

FIG. 12 is a perspective view of another embodiment of a deviceaccording to the present invention.

FIG. 13 is a perspective view of still another embodiment of a deviceaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A ligand receptor assay device according to the present inventionincludes a reaction medium having at least one reaction zone and,preferably, at least one control zone which is capable of establishingthe identity of a test subject. In a preferred embodiment of theinvention, the reaction zone includes at least one member of a ligand,ligand-receptor pair (also referred to herein as a “ligand/receptorpair”), this member being capable of establishing the presence orabsence of a substance in a test sample. In another preferred embodimentof the invention, the identity of the test subject is established byincorporating a ligand in at least one control zone to provide for atest subject identifying pattern.

A method in accordance with the invention includes conducting, on or ina portion of a reaction medium, an immunological assay for the presenceor absence of an analyte in a test sample, and separately establishingthe identity of the test subject who provided the test sample on aportion of the reaction medium.

One embodiment of the invention is directed to an assay device,preferably an immunoassay device, comprising a porous support and areaction medium covering a portion of the porous support. The reactionmedium includes at least one reaction zone and, preferably, at least onecontrol zone, wherein the control zone is capable of providing a patternsuitable for identifying a test subject. In a more preferred embodiment,the control zone is capable of providing a pattern comprising afingerprint or surface reproduction of another part of a test subject'sbody which can be used for identification. The reaction zone includes ameans for conducting a ligand/anti-ligand or ligand/ligand-receptorassay, preferably a means for conducting an assay for the presence orabsence of an analyte. In another preferred embodiment, the means forconducting an assay includes a member of an immunological pair.

Another embodiment of the invention is directed to an assay devicecomprising a reaction medium having at least one reaction zone and,preferably, at least one control zone including a member of animmunological pair, wherein the control zone is capable of establishingthe identity of a test subject. The control zone is preferably capableof providing a pattern comprising a fingerprint or surface reproductionof another part of the test subject's body which can be used foridentification.

According to another aspect, the present invention includes a support,preferably a porous support; a reaction medium, which is preferably aporous medium, mounted on the support, wherein the reaction medium hasat least one reaction zone and preferably at least one control zone, andwherein at least a portion of the control zone is capable ofestablishing the identity of the test subject. A preferred embodimentincludes a member of an immunological pair positioned in or on thereaction medium. In another preferred embodiment, at least a portion ofa control zone includes one member of an immunological pair. In someembodiments of the invention, the reaction medium may be unsupported.

The present invention is also directed to a method for processing asample comprising contacting a reaction zone of an assay device with atest sample; determining the presence or absence of at least one analytein the test sample; and forming a test subject-identifying pattern in acontrol zone of the assay device.

The present invention is also directed to a method for determining thepresence or absence of an analyte in a sample including conducting animmunoassay for the analyte in a reaction zone of an immunoassay device,whereby the presence or absence of an analyte in a test sample isdetermined; and establishing the identity of the person providing thetest sample in a control zone on the immunoassay device.

A method according to the invention includes determining the presence orabsence of an analyte in a sample, preferably by using any ligandbinding based assay, and establishing the identity of the person whoprovided the sample, preferably by using any immunologically basedprotocol. Referring to FIG. 1, the presence and/or absence of an analytein a sample may be determined by applying a biological fluid to at leastone reaction zone 20, and allowing a predetermined ligand/receptorbinding reaction to take place. In a preferred embodiment of theinvention, completion of the reaction will result in the production of asignal. Preferably, the signal is produced in the form of a visiblecolor or the absence of color. For example, in FIG. 2, the production ofvisible color hatched areas 20 ₁–20 ₃ indicates the absence of theanalyte in the test sample, and the absence of color areas (20 ₄–20 ₆)indicates the presence of the analyte in the test sample. It is intendedthat the invention should not be limited by the specific binding assayemployed, the method of producing the signal, the type of signalproduced, and the particular location of positive and/or negativeresults.

In accordance with the invention, the production of an image or patternwhich identifies the person providing the test sample may beaccomplished by applying an identifying body part, such as a person'sfinger or foot, to a control zone, and allowing a predeterminedligand/receptor reaction to take place, thereby producing the image of afingerprint or footprint. In a preferred embodiment of the invention,the tip of a person's finger, coated with a signal-producing agent,preferably a color forming agent, is applied to the surface of a controlzone having a MIP which will bond to the signal-producing agent coatedon the person's finger. In a preferred embodiment of the invention,completion of the immunological reaction will result in the productionof a visible color image which corresponds to the person's fingerprint.FIG. 2 shows an exemplary visible fingerprint.

In an embodiment of the invention, particularly for pediatric use, theimage or pattern which may be formed may be of the foot or a portion ofthe foot. In this embodiment, the foot or a portion of the foot,previously coated with a labeling reagent, may be applied to anapparatus according to the invention.

Exemplary embodiments of the invention will now be described byreference to the figures.

An immunoassay device 10 according to the invention includes a reactionmedium 14, preferably mounted on a support or base member 12. Basemember 12 is preferably a porous support. In a preferred embodiment ofthe invention, the reaction medium 14 includes at least one reactionzone 20 and at least one control zone 16. In the exemplary embodimentsshown in FIGS. 1 and 2, the immunoassay device 10 includes six reactionzones 20 (numbered 20 ₁–20 ₆) and two control zones 16 and 18.

In a detection apparatus 10 according to the invention, at least one ofthe reaction zones 20 include means for conducting an immunologicalassay, preferably to establish the presence and/or absence of an analytein a test sample. In the exemplary embodiment shown in FIG. 3, reactionzones 20 include at least one carrier, such as particles 22, embedded inreaction medium 14. As shown, the particles 22 are coated with a memberof an immunological pair 21.

In a detection apparatus 10 according to the invention, at least one ofthe control zones 16 include means for establishing the identity of atest subject, preferably including a member of an immunological pair. Inthe exemplary embodiment shown in FIG. 4, control zone 16 includes acarrier, such as particles 24, embedded in the reaction medium 14. Asshown, the particles 24 are coated with a member of an immunologicalpair 25. In a preferred embodiment of the invention, the MIP 25 iscapable of bonding to a signal-producing agent, or to aMIP-signal-producing agent conjugate.

An exemplary method according to the invention may be described byreference to FIGS. 5–8. In FIG. 5, the presence of an analyte 31 in atest sample to which a MIP 30 has been added can be determined. Thebiological fluid is applied to the surface of a reaction zone 20 havingparticles 22 coated with a member of an immunological pair 21. Ifanalyte 31 is present in the biological fluid, it will bond to or becaptured by MIP 30, with which it is mixed and incubated and which isspecific for analyte 31. The resulting immunological pair 50 will notbond to or be captured by MIP 21, and will pass through the reactionmedium 14. According to this embodiment of the invention and in thissituation, no color forming agent will bond to the coated particles 22.

In FIG. 6, the absence of an analyte in a test sample to which a MIP 30has been added can be determined. The biological fluid is applied to thesurface of a reaction zone 20 having particles 22 coated with a memberof an immunological pair 21. In the absence of analyte in the biologicalfluid, MIP 30 will bind or be captured by MIP 21. According to thisembodiment of the invention and in this situation, a color forming agentapplied to the surface of the reaction medium will bond to MIPs 30 onthe coated particles. In an alternative embodiment of the invention, theMIP 30 may include the color forming agent.

FIGS. 7 and 8 illustrate an exemplary method of producing the image of afingerprint on the surface of a control zone. The surface 60 of aperson's finger includes ridges 61 and valleys 62. When a person'sfinger is coated with a color forming reagent 102, a greater portion ofthe color forming reagent will tend to collect in the valleys 62. Whenthe finger is applied to the surface of the reaction medium 14, it isbelieved that zones of higher concentration color forming reagentlocalize in the area of the valleys. The coated particles in the surfaceof the control zone will typically bond to a higher proportion of thecolor forming reagent, which will in turn produce a more intense visiblecolor corresponding to the valley of the person's fingerprint.

In a preferred embodiment of the invention, determining thepresence/absence of a substance in a biological fluid and producing theimage of a fingerprint are typically accomplished in only a few minutes.

Both the rapidity and higher sensitivity of the test devices and methodof the present invention are attributable to characteristics of both thedevice and manner of applying reagents and/or test sample to themembrane surface and, to some extent, the materials from which themembrane is formed. Thus, as described below, the manner of applicationand materials are such that reagents in solution are maintained incontact with the membrane surface for a longer period of time to promotea greater degree of reaction between reagents in the membrane surfaceand reagents in solution. This leads to higher sensitivity and requiressmaller concentrations of reagents.

Individual aspects of the invention will now be described in greaterdetail.

Ligand-Receptor Assay

In accordance with the invention, the detection device may be used withany assay technique for detecting an analyte of interest. In all of theassays used in accordance with the present invention, an analyte isbound to at least one ligand or ligand-receptor. Exemplary bindingassays include but are not limited to labeled ligand assays, includingcompetitive binding assays in which a solid phase is coupled to thebinding reagent, such as simultaneous or sequential incubation with oneor more separation steps, and displacement assays; and labeled bindingreagent assays, including noncompetitive and competitive binding assays,including assays in which the solid phase is the binding reagent or theligand, and sandwich assays, including precipitation, radioimmunoassay,or enzyme-linked immunosorbent assay. It is intended that the inventionis not to be limited by the type of immunoassay employed or the specificprotocol used in performing the assay. Exemplary immunoassays techniquesare shown in the Examples.

Reaction Medium

In accordance with the invention, the reaction medium may be any mediumsuitable for performing a ligand-receptor assay. As noted above, thereaction medium includes at least one reaction zone to indicate thepresence or absence of an analyte and, preferably, at least one controlzone to indicate the identity of the test subject. Typically,ligand-receptor assays may be conducted using a reaction medium in theform of beads, tubes, plates, paddles, a porous or non-porous matrix, orporous or non-porous membranes or filters.

The reaction medium can include a depth medium wherein the surface or anarea near the surface comprises at least one reaction zone. The reactionmedium may also include a layered structure having a reaction zone as asurface layer. In accordance with the invention, the reaction andcontrol zones are positioned in the reaction medium so that asignal-producing agent, if present, can be readily visualized.

In accordance with the invention, the preferred reaction medium isporous, and even more preferred, a porous membrane. It is intended thatthe invention should not be limited by the type or construction of thereaction medium. One skilled in the art will readily recognize that thechoice of reaction medium depends on the desired physical properties,including, but not limited to properties such as sensitivity, bindingcapacity, stability, the type of molecules or MIPs which can be bound,and compatibility with a particular assay protocol.

Exemplary materials for forming the reaction medium include, but are notlimited to latex, nitrocellulose, cellulose, and nylon.

A preferred reaction medium is the commercially available Gelman Supormembrane. Supor membrane is a latex low protein binding polysulfonemembrane with a hydrophilic surface. One skilled in the art willrecognize that this membrane may be desirable because it providessuperior flow rate and particle retention; a smooth surface; brilliantwhiteness and opaqueness to enhance signal contrast in diagnostic tests;low extractables to reduce sample contamination and backgroundinterference; and uniform porosity to ensure final product consistency.Also, the use of this membrane obviates the need for an external wettingagent, which may be desirable for controlling the introduction ofunwanted extractables.

Another preferred membrane, IAM, Immobilon Affinity Membrane(commercially available from Millipore Corporation; see, for example,U.S. Pat. No. 4,407,943), the hydrophobic base polymer,polyvinylidenedifluoride (PVDF), binds proteins. IAM also permits a highdegree of control over the extent of protein binding and the user canreproducibly immobilize nanogram to microgram quantities of protein onthe surface to suit various assay requirements. Binding a ligand to IAMand its use in immunoassays is known in the art.

The use of membranes, as the solid support in a reaction medium of thepresent invention may be desirable for the added convenience of a solidphase, the typically high protein binding capacity, and the membrane'sflow-through characteristics. Nitrocellulose is one of the most commonlyused membranes due to its high affinity for proteins nucleic acids,other cellular antigens, and cellular macromolecules, and may bedesirable if the desired MIP binding is ionic or hydrophobic.Nitrocellulose also provides an excellent matrix for blotting proteinsand nucleic acids. The nitrocellulose may be cut into whatever shape isrequired and has the useful characteristic that the amount of protein ina fingerprint will be clearly visible.

Included within the scope of the present invention is changing orincorporating different surface properties on the membrane in order toachieve a desired result, e.g., the surface properties of a membranedesigned for a competitive binding assay for a hormone may be differentthan an immunometric assay for a therapeutic drug. For example, it hasbeen shown that treating the surface of a hydrophilized PVDF membranewith ethanolamine reduces the non-specific binding of the membranesurface. Selection of a particular surface treatment agent or surfaceproperty may be based on the desired chemical characteristic to beimparted to the surface; the inability or reduced capability ofdenaturing or impairing the functionality of a bioactive agent on or inthe reaction zone; the desire to effect a certain orientation of animmobilized bioactive molecule; the desire to promote long-termstability of an immobilized bioactive molecule; the inclusion of adesired nucleophilic substituent; and the availability and cost oftreatment agents. The use of other surface treatment agents, includingbifunctional or multifunctional reagents, to affect the surfaceproperties of the membrane are included within the present invention.

One skilled in the art will readily recognize the benefit derived byincluding a reaction medium which is porous. Positive test samples willreadily pass through the reaction medium, obviating the need to wash thesurface of the reaction medium prior to adding a color-forming agent.Although it is less desirable, it is intended that the invention includethe use of a non-porous medium. A preferred support material is asomewhat rigid porous plastic material known as Porex.

As noted above, the reaction medium may be supported or unsupported. Inthe preferred embodiment, the reaction medium is supported, and evenmore preferred, the support is porous. An exemplary support includes,but is not limited to polystyrene. In another embodiment of theinvention, the support may be impermeable to the fluids and reagentsused in conducting the testing; in this embodiment, the reaction mediumis preferably “deep” enough to have a reaction zone at or near thesurface of the medium.

In preferred embodiments of the present invention in which the device istotally or substantially self-contained, that is, needing no additionalreagents or with at most a few drops of a buffer solution or water torinse the reaction medium, and needing only a minimum level of skill toperform an accurate determination, another type of material is preferredto support the reaction medium. For such applications, a compressiblewicking or sponge-like material is preferred. Thus, by applying pressureto the outer, substantially planar surface of the reaction medium, whichis adjacent, in contact with or bonded to a substantially planar surfaceof the compressible support material, causes compression of or anindentation in the surface of the support material adjacent the reactionmedium, such that the support surface is caused to be below itsuncompressed planar surface. In the presence of a liquid, after pressureis released from the compressible support material and as thecompressible material expands to its uncompressed state, the liquid isdrawn into the support material. Thus, if a liquid sample, reagentsolution, buffer solution or water is applied to the reaction medium andsufficient pressure is applied to cause a deformation in the surface ofthe support material, liquid will be pulled into the support material asit expands to its original state. The pressure applied is a function ofthe compressibility of the material. A preferred compressible supportmaterial is a compressible, flexible reticulated urethane foam,preferably a polyester or polyether urethane foam having a pore ratingof about 75 to 100 ppi, preferably about 80 to 90 ppi. Such materialsare available from E. M. Murray Co. Inc.

Control Zone

In a preferred embodiment of the invention, the reaction medium includesa control zone for establishing a reference point in determining thepresence or absence of an analyte in a test sample (reference controlzone), and most importantly a control zone for establishing the identityof the test subject who provided the test sample (identity controlzone).

In accordance with the invention, a control zone for establishing theidentity of the test subject (identity control zone) who provided thetest sample may include any signaling mechanism which incorporates aMIP. For example, the control zone may include a member of animmunological pair capable of bonding to a signal-producing agent, whichwhen applied by contacting the test subject's finger to the surface ofthe identity control zone, an image of the fingerprint is produced onthe test device.

As used herein, “capable of establishing the identity of a test subject”refers to the capability of specifically identifying the subject whoprovided the sample being tested. In a preferred embodiment of theinvention, the control zone includes a member of an immunological pairwhich, when placed in contact with the test subject's finger which hasbeen previously coated with a signal-producing agent, is capable ofproducing a permanent image of a fingerprint, preferably a fingerprintimage easily visible. In another embodiment, the invention includes acontrol zone having a member of an immunological pair or nucleotidesequences which are capable of bonding to nucleotide sequences which canbe used to identify the test subject.

In accordance with the invention, the control zone for establishing areference point in determining the presence or absence of an analyte ina test sample includes any means for providing a control. Severalexamples for providing a control are shown in the examples. It isintended that the invention not be construed as being limited by themeans or mechanism for establishing the standard of comparison injudging the test results.

Reaction Zone

The reaction zone refers to the locus in which the presence or absenceof an analyte can be determined. For example, the reaction zone ispreferably on or near the surface of the reaction medium, or may bebelow the surface. In a preferred embodiment of the invention, thereaction zone includes at least one ligand or ligand-receptor bound to aparticle or bead in a matrix or filter, suitable for capturing ananalyte of interest. In another embodiment of the invention, at leastone of the reaction zones may be a control for establishing a referencepoint, e.g., a comparative control to show the absence of color.

Signal-Producing AGENT

A signal-producing agent refers to any agent or marker which produces adetectable signal or which permits the detection of a ligand orligand-receptor. Preferred signal-producing agents are those whichpermit detection of the analyte without instruments, preferably byvisual means. Exemplary signal-producing agents include, but are notlimited to color forming agents, such as an enzyme, polymer containingdyes, chemiluminescent agents, fluorescent agents, radioisotopes, orferromagnetic particles. The color forming agent may be a coloredparticle, a colored molecule or some species, such as an enzyme, whichis capable of triggering a sequence of events leading to the formationof a colored marker. The colored molecule may be a fluorescent dye, suchas fluorescein or rhodamine; a chemiluminescent compound; abioluminescent compound; or a compound that may be detected by theabsorption of electromagnetic radiation (and possible reemission ofradiation at another wavelength), including ultraviolet radiation,visible radiation and infrared radiation. The colored molecule may bedirectly or indirectly conjugated to a ligand or ligand-receptor.Alternatively, the colored molecule may be incorporated in a particle,particularly a microsome.

Enzymes, useful as color forming agents, include alkaline phosphatase,horseradish peroxidase or B-galactosidase. Such enzymes are often usedin conjunction with a chromogenic substrate. A list of some exemplarychromogenic substrates which may be used with enzyme color formingagents are given below in Example 11.

In a preferred embodiment of the invention the color forming agent maybe an electron dense particle, such as colloidal gold, silver coatedcolloidal gold or ferritin.

In another preferred embodiment of the invention, the color formingagent is capable of bonding to a MIP.

The present invention includes a signal-producing agent, which may be acolor forming agent. Preferably, the signal-producing agent comprises alabeled MIP, for detecting the adsorption of an analyte or an analyteconjugate in or on the reaction medium. The labeled MIP may comprise anyone of a number of appropriate labels, such as an enzyme, a fluorescentcompound, a bioluminescent compound, a ferromagnetic atom or a coloredparticle or microsome. In a preferred embodiment, the label used in thepresent invention is colloidal gold. Gold is biologically inert, hasvery good charge distribution and is widely available in many usefulforms. Its detection can be enhanced using several silver depositionmethods, which permit development to be monitored by the naked eye.Colloidal gold particles conjugated with a wide range ofanti-immunoglobulin antibodies, protein A or streptavidin are availablecommercially.

While a colloidal gold substrate is preferred over other dyed particlesor microsomes, a chromogenic substrate provides an alternate sensitivedetection method for the enzyme conjugate.

Several embodiments of the present invention relate to compact assaydevices and kits which contain all or most of the reagents and materialsnecessary for qualitative and/or quantitative determinations of one ormore analytes. A preferred embodiment of such a device is illustrated inFIG. 11 which shows a self-contained assay device 110. The deviceincludes a housing 111 which in a preferred embodiment includes a firstpart 111 a and a second part 111 b. Located within the first part of thehousing 111 a, mounted on the upper surface of a raised plateau orplatform portion of a base member 112 is a reaction medium 114, of thetype described above. The base member 112 preferably includes a poroussupport adjacent, in contact with or bonded to the reaction medium 114.In a preferred embodiment of the invention, the reaction medium 114includes one or more reaction zones 120, each of which includes aspecific reagent, such as a member of a ligand/receptor pair (which maybe a member of an immunological pair, for example an antigen or anantibody). The reaction medium in this preferred embodiment alsoincludes at least one control zone 116. As in the embodiments discussedabove, each of the reaction zones includes a reagent suitable fordetermining the qualitative presence or the quantitative range of ananalyte. In a preferred embodiment of the invention, a control zone issuitable for identifying the donor of a test sample. Likewise, thecontrol zone 116 also includes a suitable test reagent for identifyingthe test subject which is preferably a member of a ligand/receptor pair,such as a member of an immunological pair.

The reagents present in each of the reaction zones may be appliedmanually or, preferably, using an automated process, such as using anink jet sprayer of the type used in ink jet printing systems, to applythe reagent in individual and segregated reaction zones.

In the second part of the housing 111 b, is located a medium 126 onwhich is spread at least one signal-producing agent, oranalyte-indicating agent, which is preferably a color forming agent. Thesignal-producing agent-containing medium 126 is formed from a materialwhich is of a lower porosity than either the reaction medium or themedium for containing a test sample (discussed below). Preferable foruse as the medium on which the signal-producing agent is spread is aclosed cell polyolefin material, such as a closed cell polyethylene orpolypropylene. A preferred material is available as Minicell from VoltekCorporation. The signal-producing agent, as with the other embodimentsof the present invention, preferably is a labelled member of aligand/receptor pair, such as a labelled member of an immunologicalpair, such as a colloidal gold labelled substance. The medium 126,containing the signal-producing agent, is positioned in the second partof the housing 111 b in such a way that when the two parts of housingare brought together in a specific closed position, contact is madebetween the medium 126 and the reaction medium 116 such thatsignal-producing material contained in the medium 126 is transferred tothe reaction medium. This is preferably accomplished by mounting themedium 126 on a base portion 128 which resembles an elevated plateau orplatform, which in the preferred embodiment shown in FIG. 11 has a shapesimilar to base 112. This arrangement, in which the medium containingthe signal-producing agent is spaced apart from the reaction medium 114in a first or open position and transfers the signal-producing agent tothe reaction medium 114 in a second or specific closed position,apparently achieves greater sensitivity and increased speed due to thepressure applied when the signal-producing agent-containing medium 126contacts the reagent medium 114. Depending upon the specific materialsinvolved, a pressure in excess of atmospheric may be used, preferablyslightly less than about 2 to about 5 psi, for a period of about 5 toabout 20 seconds is sufficient to adequately retain the signal-producingagent at or near the surface of the reaction medium after the media 114and 126 are placed in contact with one another. Although higherpressures can generally be used to transfer either the signal-producingagent, analyte or conjugate formed of the analyte with a ligand receptorof the analyte (as discussed below), the duration of time for exertingthe pressure is decreased, since pressure and time vary inversely.

Another component of the self-contained or “integrated” deviceillustrated in FIG. 11 is a medium 185 for containing and transferring aportion of a test sample to the reaction medium 114. The medium 185 usedto hold both a test sample and a reagent capable of reacting with ananalyte is a hydrophilic material, preferably in the form of ahydrophilic sponge and preferably is formed from a fine pored polyesterurethane sponge material. A preferred material is polyester urethanefoam sponge material having a porosity of 100 ppi that is available fromE. N. Murray Company Inc. The medium 185 serves several purposes. First,it is capable of absorbing a test sample taken from a donor or testsubject. It is also capable of transferring the sample or a conjugateformed from an analyte present in the test sample and a receptor againstthe analyte to the reaction medium 114. The medium 185 is also capableof providing diffusion and reaction loci for both the substances presentin the test sample, as well as a reagent present in the medium. Thisreagent is preferably a member of a ligand/receptor pair, such as amember of an immunological pair of the type discussed above andidentified in FIGS. 5 and 6 as MIP 30. The reagent in the medium 185 istypically an antibody. This reagent is contained in the medium 185preferably as a lyophilized antibody. In this embodiment, the spongemedium 185 is treated with a solution of reagent and the treated spongeis subjected to a lyophilization treatment in a lyophilization chamber,such as a Speed-Vac.

The medium for containing the test sample and the test sample and thereagent, preferably in lyophilized form, may be mounted at one end of aremovable, cylindrical member 140. In the embodiment illustrated in FIG.11, the open end 140 a opposite to the closed end 140 b of thecylindrical member 140 which the medium 185 is mounted, is larger inshape or diameter than the base portion 128 of the housing part 111 b onwhich the signal-producing agent-containing medium 126 is mounted.Preferably the open end 140 a of the cylindrical member 140 iscommensurate in shape to the base portion 128 so that the cylindricalmember 140 may be easily accommodated in the housing part 111 b over thebase portion 128. The cylindrical member 140 is of such a shape andconfiguration that in an “open” position of the housing parts in whichthe media 114 and 126 are in spaced-apart relationship with one another,the medium 185 is also in spaced-apart relationship with both of themedia 114 and 126 in the open or first position of the housing members111 a and 111 b. In a second or specific “closed” position of thehousing members 111 a and 111 b, while remaining within the housing 111and in spaced relationship to the base 128 and signal-producingagent-containing medium 126, the medium 185 for containing the samplecontacts and transfers substances present in the sample and/or aligand/receptor pair formed between an analyte found in the sample and aconjugate thereof to the reaction medium 114. Thus, in the second orclosed position, pressure is exerted by the end 140 b of the cylindricalmember and medium 185 on the reaction medium 114 in much the same manneras in the contact which occurs between the medium 126 containing thesignal-producing agent and the reaction medium in a second closedposition. The method of using this device is discussed below.

While FIG. 11 illustrates an embodiment of the present invention whichemploys a two-part housing 111 having a first part 111 a and a secondpart 111 b, and a removable, cylindrical member 140 on which a sponge185 for containing a test sample is attached, other aspects andalternative embodiments are contemplated by the present invention. Forexample the housing parts 111 a and 111 b are illustrated in FIG. 11 asbeing joined to each other by a flexible hinge arrangement. When thehousing is formed from a suitable plastic, which is preferablysubstantially inert to any of the reagents or materials used in othercomponents of the device or in solutions applied to the media of thedevice, the hinge may be formed from a thinner portion of the sameplastic material. Thus, the housing may be formed as a single unit inone pressing operation. Alternatively, another material may be used asthe hinge material and the hinge attached to the housing parts bysuitable means.

In another embodiment, the housing parts 111 a and 111 b need not bejoined to one another at all. Thus, to close the device, the separateparts may be constructed to snap into place with a detent or somesimilar arrangement or may engage one another with a threadedarrangement or a bayonet engaging arrangement.

In yet another alternate embodiment, the medium 185 for containing thesample and containing a test reagent, rather than being mounted on theremovable, cylindrical member 140 of the type shown in FIG. 11, may belocated in a third housing part. When used with housing parts which arepermanently joined to one another, such as that described immediatelyabove, the housing part containing the sponge for containing a testsample may be placed intermediate the separate housing parts containingthe reaction medium and the signal-producing agent-containing medium(equivalent to housing parts 111 a and 111 b). In this instance,however, the intermediate housing part containing the sponge 185 mayengage one or both of the other housing parts and contact the reactionmedium in the first housing part when in place and forming part of thehousing. It may also be removed when it is desired to contact thereaction medium 114 with the medium containing a signal-producing agent126.

In yet another embodiment, the housing part containing the hydrophilicsponge for containing a test sample may be joined to the first housingpart (equivalent to housing part 111 a as shown in FIG. 11 by means of ahinge on the edge portion of the first housing part in opposingrelationship to the edge of the first housing part to which the secondhousing part is joined. Thus, when it is desired to place the spongecontaining a test sample in contact with the reaction medium, the thirdhousing part containing the sponge may be rotated to asubstance-transferring position in contact with the reaction medium andthereafter rotated to a spaced apart position with respect to thereaction medium and the second part of the housing containing thesignal-producing agent medium 126 may be rotated into transferablecontact with the reaction medium.

The above embodiments of the present invention which include three media(a reaction medium, a medium for containing a test sample, and a mediumcontaining a signal-producing agent) are substantially self-contained.Thus, it is unnecessary to add additional reagents in order to performthe assay. At most, a buffer solution, such as a phosphate bufferedsolution, or water may be used in a final step to rinse the reactionmedium to remove residual reagents from the surface of the reagentmedium. Alternatively, when a compressible porous medium is used as asupport for the reaction medium, it may be unnecessary to use anysupplemental materials.

The present invention also contemplates compact assay devices using onlytwo media, rather than the three-media devices discussed above. Thus, inthe embodiment illustrated in FIG. 12, while still retaining thereaction medium 114 mounted on base 112 in a first part of the housing,the sample-containing and transferring sponge 185 may be mounteddirectly on the base 128 instead of the signal-producingagent-containing medium 126 being located thereat. In such anembodiment, the removable, cylindrical member 140 on which the medium185 is mounted in the “3-media” embodiment illustrated in FIG. 11 may bedispensed with. In such an embodiment, the wash bottle containing abuffer, detergent or water may be used in the same manner as describedabove. However, in this embodiment, the signal-producing agent may bedispensed from a reagent bottle directly on the finger or otheridentifying body part of the test subject and the finger or other bodypart may be placed in contact with the reaction medium after the testsample has been placed on the sponge 185 mounted in base 128, incubationhas taken place and the housing has been closed such that there issubstance transferable contact with suitable pressure between thesurface of sponge 185 and the reaction medium 114.

Another two-media device is illustrated in FIG. 13. Like the embodimentdiscussed above and shown in FIG. 11, this includes the reaction medium114 mounted on base 112 in a first part of the housing and thesignal-producing agent-containing medium 126 in the second part of thehousing. This corresponds substantially to the embodiment shown in FIG.11 without the removable, cylindrical member 140 and medium 185 forcontaining a test sample. This embodiment, like the 3-media deviceillustrated in FIG. 11 does not require the presence of the testsubject, and, accordingly, will not produce a fingerprint or otheridentifying mark of the test subject when the test subject is notpresent. Alternatively, however, a fingerprint can be produced when thetest subject is available at the time the test is performed. In thisembodiment, the sample is mixed with a test reagent supplied with thedevice, most suitably in a reagent bottle, as part of a kit. The reagentmay include one or more substances capable of indicating the presence ofone or more analytes and is preferably a member of a ligand/receptorpair, most preferably a member of an immunological pair capable offorming a conjugate with a specific analyte. The mixture of test sampleand reagent is applied dropwise to the surface of the reaction medium,sufficient time for incubation allowed and the housing is closed for ashort period of time so that there is contact between thesignal-producing agent-containing medium 126 and the reaction mediumsufficient to transfer the signal-producing agent to the surface of thereaction medium. If the test subject is present at the time of testing,rather than closing the device to make contact between the media 114 and126, the test subject may roll a finger on the surface of thesignal-producing agent medium 126 and then press the finger on thereaction medium.

Another embodiment of the present invention is directed to a device andmethod for distinguishing between an acute and a chronic drug abuser.Thus, in the body of one who uses a drug, not only are molecules of thedrug or a metabolite found, but the drug or such metabolite binds to acarrier molecule. Continued use of a drug stimulates production of anantibody against the carrier bound drug. Thus, in the body of a humanwho chronically uses a drug may be found not only a residue of the drugor metabolite thereof, but also the presence of a human antibody to thedrug. In an embodiment of the device according to the present invention,a reaction zone in the reaction medium may be spotted with some of thedrug of which the test subject is suspected of using. This will thenform a conjugate with the human antibody to the drug if present as ananalyte in a test sample. As a signal-producing agent for indicating thepresence of the drug antibody may be used the corresponding goat ormouse antihuman labelled antibody.

Method of Use

An assay method in accordance with the invention includes conducting aligand-receptor assay for an analyte in a reaction zone of an assaydevice, whereby the presence or absence of an analyte in the test sampleis determined, and establishing the identity of the person providing thetest sample in a control zone on the assay device. In a preferredembodiment of the invention, establishing the identity of the personproviding the test sample may be accomplished using ligand-receptorassay methods, e.g., binding a MIP to a signal-producing agent.

As noted above, the biological fluid sample to be tested for thepresence or absence of at least one analyte is applied to the surface ofthe immunoassay device according to the invention. Any means of applyingthe fluid sample to the detection apparatus may be used. For example, inone embodiment of the invention, the biological fluid, e.g., urine, maybe combined with a MIP according to standard immunoassay procedures,drawn into a pipette, and deposited on the surface of the test device.

In another example, the biological fluid, e.g., blood, may be collectedin a pipette and deposited on the surface of the test device, or may besmeared on a solid applicator, such as a fingertip. In one preferredembodiment, blood is smeared on the test subject's fingertip, which isthen pressed in contact with the surface of the detection device. Inanother preferred embodiment, the biological fluid is combined with aMIP before application to the reaction medium. In still anotherpreferred embodiment, the sample is deposited on the surface of a mediumfor containing a test sample. Other exemplary methods of applying thetest sample to the detection device are shown in the Examples.

A preferred embodiment of the invention includes applying one or morereagents or a sample to a surface of the test device using pressure. Forexample, it has been found that smearing a signal-producing agent on thetest subject's fingertip and applying the fingertip to the test devicesignificantly reduces the amount of signal-producing agent required, andsignificantly increases the sensitivity and speed at which the assayproceeds. For example, as little as 10 microliters and preferably about12 microliters of colloidal gold can be smeared on the test subject'sfingertip in order to perform the assay. In contrast, as much as 100microliters or more of the signal-producing agent is typically requiredfor conventional flow-through cassette assay devices. In a like manner,another preferred embodiment includes the use of pressure in theapplication of sample to the surface of the reaction medium.

While not intending to be limited to a particular theory of operation,it is believed that the pressure and/or surface tension which occurswhen the test subject's finger is placed on the assay device results inlocalizing an increased concentration of ligand and ligand receptor,increasing the amount of time that the reagents are retained in alocalized area of the assay device, and may involve migration of thesignal-producing agent from the ridges of the fingerprint to thevalleys, to produce a clear fingerprint pattern on the surface of thetest device.

The higher sensitivity and reduced amounts of reagents provided by thepresent invention seems to result from a more complete reaction takingplace at the surface of the membrane where immobilized reagent is found.The more complete reaction results apparently from the reagents appliedto the surface of the membrane in liquid form being maintained thereatfor a longer period of time. Thus, as is typical with most known testdevices, liquid samples and/or reagents are applied in drop form to thesurface of a membrane which contains either reagent or test sample andare quickly drawn through the membrane, either because of an absorbentplaced in contact with the membrane on its downstream surface or becauseof the nature of the membrane material. In the present invention,however, the liquid containing the reagent and/or sample is applied tothe surface of the reaction medium and is maintained thereat with theapplication of pressure greater than atmospheric pressure as a thin filmwhich shows little tendency to pass completely through the membrane.Thus, using the finger as an applicator or another applicator means,such as an applicator means included as part of the device,reagent-containing liquid on the applicator is transferred to andmaintained at the upstream surface of the reaction medium underpressure. Thus, while not wishing to be held to any theory, it appearsthat primarily the surface tension of the liquid, in combination withother factors such as the capillary action of the applicator andpossibly the porosity of the medium on which the liquid is placed incombination with the pressure causes a thin film of liquid to bemaintained at the upstream surface rather than passing through themedium as long as the pressure is maintained on the outer surface of themedium. The contribution of the surface tension in maintaining a thinfilm at the surface of the reaction medium appears to increase theavailability of the substance(s) present in the liquid to reagents inthe medium. Thus, using an applicator, such as the finger, with apressure greater than atmospheric pressure, typically as little as about1 to about 5 psi and up to a pressure that a test subject would normallyand comfortably apply (such as in pressing the finger in making an“official fingerprint”), prevents the liquid from passing through themembrane until the pressure is removed. Application of pressure wouldnormally be for a period of time of about 5 to 20 seconds.

Maintaining the reagent at the surface of the medium or membrane forthis period of time is sufficient to assure a greater degree of reactionof the reagents located at or immediately below the upstream surface ofthe medium. While the nature of the liquid, such as its surface tension,as well as the materials from which the membrane and applicator areformed, have some bearing on this phenomenon, it appears that a varietyof materials may be used and the phenomenon of maintaining the thin filmof liquid at the surface of the membrane still occurs. Furthermore, andalso not wishing to be held to a particular theory, it appears that theincreased sensitivity and particularly the rapidity of the assaydeterminations of the devices and methods of the present invention maybe attributed, at least in part to the use of pressure at the timeliquid solutions are applied to the surface of the reaction medium. Itis believed that the pressure applied, which is greater than atmosphericpressure and is typically as little as 1 psi and preferably in the rangeof about 2 to 15 psi, increases the kinetics or the speed of theparticular reaction taking place.

The test device preferably includes one or more MIPs immobilized in acorresponding number of reaction zones. For example, if the analyte ofinterest is an antibody, the test device may include an antigen bound tomicrospheres or particles embedded in the reaction zone. One skilled inthe art will recognize that a desired amount of antigen can beimmobilized in the reaction zone, and that a threshold concentration ofbound MIP may be used to detect a predetermined amount of analyte. Inone embodiment of the invention, the detection device includes severalreaction zones, each reaction zone for a different analyte, and eachreaction zone includes a predetermined threshold concentration of boundMIP to detect a predetermined amount of analyte. As used herein,threshold amount or concentration refers to the lower limit of aconcentration range for an analyte. In another embodiment of theinvention the detection device includes several reaction zones, eachreaction zone including a different threshold concentration for the sameanalyte. In this embodiment, the threshold concentration provides areference point for determining the upper limit of an analyteconcentration range. As shown in the examples below, varying thethreshold concentration in the reaction zones may provide an exemplarymethod of quantifying the amount of analyte in a sample.

After the biological fluid being tested has been applied to the surfaceof the detection device, the presence or absence of the analyte in thetest sample can be determined, preferably by visually ascertaining acolor (signal) or the absence of a color (signal). In a preferredembodiment of the invention, a positive response, i.e., indicating thepresence of the analyte in the biological fluid, corresponds to theabsence of color. In a preferred embodiment of the invention, a negativeresponse, i.e., indicating the absence of the analyte in the biologicalfluid, corresponds to a visualized color. However, in one embodiment ofthe invention in which a determination is made as to whether the testsubject is a chronic drug abuser in which a human antibody to a drug isbeing tested for and the reagent present in the reaction zone is thedrug itself and the labelled signal-producing agent is, for example, agold labelled goat or mouse antihuman antibody, a positive response,indicating the presence of the drug antibody, corresponds to theappearance of color.

In accordance with one aspect of the invention, when the biologicalfluid being tested is blood, it has been unexpectedly found that wholeblood may be directly applied to the surface of the test device withoutthe need for serum separation prior to the sample being applied to thetest device. In accordance with this embodiment of the invention, thewhole blood sample is applied to the surface of the test device, andthen a detergent is applied over the blood sample. While it is knownthat detergents such as TWEEN may be used to remove red cells and thelike from whole blood samples, it has been unexpectedly found that theapplication of a clearing reagent, such as a detergent (preferably adetergent which includes a mixture of ionic, and nonionic detergents,and an alcohol), allows the application of whole blood to the surface ofthe assay device without the need to separate the cellular componentfrom the serum prior to application to the device. The application of adetergent is preferable because the detergent disrupts blood cells. Theruptured cell debris then pass through the medium and are removed fromthe reaction zone, i.e., removed from interfering with immobilizedsurface antigens. Once the presence or absence of the analyte has beendetermined, the detection device made in accordance with the presentinvention may be used to identify the test subject whose biologicalfluid was tested. In a preferred embodiment of the invention,determining the identity of the test subject includes performing aligand-receptor assay which results in the development of a fingerprintof the individual. In accordance with a preferred embodiment of theinvention, determining the identity of the test subject is independentof any reactions with the analyte in the fluid.

In accordance with this method of the invention, the person's fingertipis coated with a signal-producing agent conjugated to a MIP. When thecoated fingertip is applied to the surface of a control zone, thesignal-producing conjugate binds to a MIP embedded in the control zoneof the test device. While the invention is not to be limited to aparticular theory of operation, it is believed that the ridges andvalleys of a person's fingertip provide localized areas of concentratedsignal-producing conjugate (corresponding to a valleys) and localizedareas of low concentration signal-producing agent (corresponding to aridge). When applied to the surface of a control zone of a deviceaccording to the invention, a clearly identifiable image of afingerprint is produced, whereby the identity of the person providingthe test sample can be provided.

In accordance with the invention, the presence or absence of an analyteof interest in a sample may be determined by applying the sample to thesurface of a reaction medium, the reaction medium including at least onereaction zone having a member of an immunological pair therein; andapplying a signal-producing agent which binds, directly or indirectly(e.g., a secondary antibody), to the MIP or to the analyte of interest.

The self-contained test device illustrated in FIG. 11 may be used asfollows:

A test sample in liquid form, such as a bodily fluid (for example,saliva, urine or blood) is placed on the outer surface of the medium 185for containing and transferring the test sample. Up to about 20 drops oftest sample may be applied to the medium 185. Since this device providesso much greater sensitivity and speed than do conventional test devices,as little as one drop of test sample may be employed preferably,however, about 3 to about 10 drops of test sample are preferably used.With the removable, cylindrical member 140 in place in the housingdisposed with the open end thereof over the base 128 andsignal-producing medium 126 in the first part of the housing 111 b, thetest sample is applied to the outer surface of the medium 185 or thesample may be applied to the medium and the cylindrical member 140thereafter placed in housing part 111 b. Any analyte which is present inthe test sample undergoes reaction with a member of a ligand/receptorpair, preferably a member of an immunological pair, present inlyophilized form in the sponge 185, to form a conjugate. A period ofabout 1 to about 4 minutes, preferably about 2 minutes, is permitted forincubation (reaction) of the test sample and member of theligand/receptor pair. With the cylindrical member 140 remaining in placein the second part 111 b of he housing, the two parts of housing 111 arebrought together to a first closed position in which the outer surfaceof the sponge 185 contacts the reaction medium 114 and exerts sufficientpressure to transfer substances present in the test sample, the memberof the ligand/receptor pair and any conjugate formed between the analyteand the member of the ligand/receptor pair. In this position, althoughbeing referred to as a “closed position,” the housing may not beentirely closed, depending on the particular structure employed. Thisclosed position is maintained for a period of about 1 to about 4minutes, preferably about 2 minutes. Thereafter, the housing is openedand the cylindrical member 140, containing the sponge 185 is removedfrom the housing device. The device is then closed to a fully closed orsecond closed position in which the signal-producing agent-containingmedium 126 contacts the reaction medium 116 with a sufficient pressureto transfer the signal-producing agent, such as a gold-labelled memberof a ligand/receptor pair to the reaction medium. The housing is!maintained in this second or fully closed position for a period of atleast about 5 to about 30 seconds, preferably about 15 seconds.

After about a minute the device will indicate the presence of anyanalytes being tested for, or an amount of analyte being present inexcess of a predetermined concentration or amount, by showing a blankspace in the appropriate reaction zone. In most applications, a negativetest is reflected by a coloration of the appropriate reaction zone whena specific analyte is absent or is present in amounts whereconcentration is below a predetermined value. A device will not providean indication of the identity of the test subject at this stage unlessthe test subject places a finger or other identifying portion of thebody in contact with the control zone 116 of the reaction medium 114after opening the housing for the second time. Alternatively, ratherthan applying the finger or other body part to the control zone of thereaction medium after contact has been made between the medium 126 and114, this step of pressing the two media together may be omitted and thefinger or other body part of the test subject may contact under pressurethe reaction medium after first being rolled over the signal-producingagent-containing medium 126 or applying the signal-producing agentdirectly to the finger or other body part with an applicator ordispenser. While each of these methods of identifying the test subjectby a permanent and individual characteristic of the test subject is bothrapid and sensitive (the total determination typically taking no morethan about 3 to 5 minutes), the last described method in which thesignal-producing agent is applied directly to the fingertip which isthen applied to the reaction medium, is somewhat more effective in thatsmaller amounts of reagent and sample may be required and the speed ofthe process is somewhat more rapid. The method in which a finger orother identifying body part is not placed in contact with the reactionmedium is least effective in terms of the amounts of material requiredand speed of the process while contact between the finger or other bodypart with the transfer medium is intermediate in effectiveness.

Test Kit

In an embodiment of the invention, the assay device made in accordancewith the invention may be incorporated into a kit. The kit may alsoinclude any of a number of reagents for performing the ligand/receptorassays. For example, the kit may include a test device, at least oneagent for producing a signal (or an “ink” pad for transferring thesignal-producing agent to the finger), and one or more washing, clearingor detergent reagents. The kit may also include one or more ligands orligand receptors, such as lyophilized primary antibody and a secondaryantibody, and one or more buffers. The ligand or ligand receptor may bea labelled material.

When the compact devices are supplied in kit form, they may beaccompanied by a wash bottle containing a phosphate buffer solution.Such wash bottle and/or reagent bottle may be supplied most effectivelyas a kit with the alternate two-media embodiments of the presentinvention shown in FIGS. 12 and 13, discussed above. Thus, rather thanthe device illustrated in FIG. 11 containing a housing and the threemedia, 114, 126 and 185, a device including a housing and two of thethree media, and appropriate reagents contained therein may be employed.

SPECIFIC EXAMPLES Example 1

One embodiment of a immunoassay device of the present invention wasprepared according to the following procedure. A reaction zone wasconstructed by embedding polystyrene particles, coated with human serumalbumin-benzoylecgonine (HSA-BE), in one portion of a low proteinbinding polysulfone membrane (Gelman Supor membrane) mounted on a poroussupport. A control zone 16 was constructed by embedding polystyreneparticles, coated with goat anti-mouse antibody, in a different portionof the membrane. A representative schematic cross section of thereaction and control zones is shown in FIGS. 3 and 4.

The assay device was prepared according to the following procedure. Thereaction medium was first rinsed with a blocking buffer solution(Solution A) consisting of 2% polyvinyl alcohol (PVA), 1% glycine and0.05% TWEEN-20 (polyoxyethylenesorbitan monolaurate) in a phosphatebuffered saline solution. The central well of the immunoassay device(control zone 16) was spotted with a dilute solution of polystyrenelatex coated with goat anti-mouse immunoglobulin G (adsorbed againsthuman serum) in a phosphate buffered saline containing 4% sucrose, 1.0%BSA and 0.05% azide. The reaction zone was spotted with a dilutesolution of polystyrene latex coated with human serumalbumin-benzoylecgonine (HSA-BE) in 0.2 M sodium bicarbonate. Thereaction medium was then inverted onto hydrophobic polyethylene anddried for one hour in a drying room set between 80 and 100° F. Afterremoval of the reaction medium from the hydrophobic polyethylene, theassay device was ready for use.

Example 2

A saliva sample was obtained from a person to be tested for the presenceor absence of benzoylecgonine. A 200 microliter aliquot of a dilutesolution of mouse anti-benzoylecgonine immunoglobulin G (mouse anti-BEIgG) in a phosphate buffered saline, which contained 0.1% bovine serumalbumin (BSA) and 0.05% TWEEN-20, was added to a 200 microliter aliquotof the saliva sample. The resulting mixture was allowed to incubate forthree minutes. During this incubation period, 400 microliters ofSolution A was added to the reaction medium of the immunoassay deviceprepared in Example 1 and allowed to drain through the reaction medium.The mouse anti-BE IgG/saliva mixture was then added to the reactionmedium and allowed to incubate for two minutes. Another 400 microliteraliquot of Solution A was then added to the reaction medium and allowedto drain. After the Solution A had finished draining, a finger of theperson who had provided the saliva sample was painted with 15microliters of a dilute solution of colloidal gold conjugated goatanti-mouse immunoglobulin G in a TRIS buffer containing 1.0% BSA, 0.05%TWEEN-20 and 0.05% azide (hereinafter “Gold Label Solution”). The fingerwas gently pressed against the reaction medium of the immunoassay deviceand held in place for three seconds. The finger was then carefullyrolled off the reaction medium. The reaction medium was allowed toincubate for fifteen seconds before another 400 microliter aliquot ofSolution A was added to the reaction medium. After completion of theprocedure, the reaction zone was colored, showing a negative test resultfor benzoylecgonine (e.g., as shown with reaction zones 20 ₁–20 ₃ inFIG. 2).

The determination was repeated except that a minor amount ofbenzoylecgonine was added to the saliva sample just prior to theaddition of the mouse anti-BE IgG solution. In this instance, aftercompletion of the test, the reaction zones were completely white (e.g.,as shown with reaction zones 20 ₄–20 ₆ in FIG. 2). At the completion ofboth determinations, the central well control zone contained an imprintof the finger of the person who had provided the saliva sample (e.g., asshown with the fingerprint 62 on control zone 16 in FIG. 2).

Example 3

An immunoassay device is prepared according to the procedure of Example1 except that the device contains six reaction zones for differentanalytes. Each reaction zone contains polystyrene particles coated witha different analyte conjugate embedded in the membrane. Reaction zones20 ₁–20 ₆ contain polystrene latexes coated respectively with cocaine(20 ₁), opiates (20 ₂), PCP (20 ₃), amphetamine/methamphetamine (20 ₄),tetrahydrocannabinol (20 ₅) and alcohol (20 ₆) as the analytes (as shownin FIG. 1). The central control zone 16 is prepared as in Example 1 withpolystyrene particles, coated with goat anti-mouse immunoglobulin G,embedded in the membrane.

A saliva sample is obtained from a person to be tested for the presenceor absence of the six analytes. A 200 microliter aliquot of a dilutesolution of mouse anti-analyte immunoglobulin G for each of the sixdifferent analytes in a phosphate buffered saline (hereinafter “mouseanti-analyte IgG Solution I”) is added to 200 microliters of the salivasample. As described in Example 2, while this mixture is incubating, thereaction medium is pretreated with Solution A. After the mouseanti-analyte IgG Solution I/saliva mixture is added to the reactionmedium, the remainder of the test procedure described in Example 2 isfollowed. The reaction medium is treated with Solution A, contacted witha finger of the test subject that has been coated with a dilute solutionof colloidal gold conjugated goat anti-mouse immunoglobulin G and thentreated again with Solution A. After this procedure is carried out,central control zone 16 contains the imprint of the finger of the personproviding the saliva sample. Reaction zones 20 ₁–20 ₆ are all colored,showing a negative test result for the presence of all six analytes.

The remaining saliva is then spiked with minor amounts ofamphetamine/methamphetamine, tetrahydrocannabinol and alcohol. A 200microliter aliquot of mouse anti-analyte IgG Solution I is then added to200 microliters of the spiked saliva and the determination is repeatedas described for the unspiked sample. After the procedure is carriedout, the assay device exhibits the result shown in FIG. 2. Centralcontrol zone 16 contains the imprint of the finger of the personproviding the saliva sample. Reaction zones 20 ₁–20 ₃ show a negativetest result and are colored. Reaction zones 20 ₁–20 ₆ show a positiveresult and are white.

Example 4

An immunoassay device, containing six reaction zones, is preparedaccording to the procedure of Example 1. As in Example 3, reaction zones20 ₁–20 ₆ respectively contain polystrene latexes coated respectivelywith cocaine (20 ₁), opiates (20 ₂), PCP (20 ₃),aphetamine/methamphetamine (20 ₄), tetrahydrocannabinol (20 ₅) andalcohol (20 ₆) as the analytes. In this instance, however, centralcontrol zone 16, prepared as in Example 1 contains polystyrene particlescoated with goat anti-mouse immunoglobulin G embedded in the membrane. Aurine sample is obtained from a person to be tested for the presence orabsence of the six analytes. A 200 microliter aliquot of the urine ismixed with 200 microliters of mouse anti-analyte IgG Solution I and thetest procedure is then carried out exactly as described in Example 2.After completion of the determination, the central control zone 16contains the imprint of the finger of the person providing the urinesample and reaction zones 20 ₁–20 ₆ are all colored showing a negativetest result for the presence of all six analytes.

The remaining urine is then spiked with minor amounts ofamphetamine/methamphetamine, tetrahydrocannabinol and alcohol. A 200microliter aliquot of mouse anti-analyte IgG Solution I is then added to200 microliters of the spiked urine and the determination is repeated asdescribed for the unspiked sample. After completion of thedetermination, the immunoassay device exhibits the result shown in FIG.2. Central control zone 16 contains the imprint of the finger of theperson providing the urine sample. Reaction zones 20 ₁–20 ₃ are colored(negative result) and reaction zones 20 ₄–20 ₆ are white (positiveresult).

Example 5

The reaction medium 83 of the immunoassay device shown in FIGS. 9 and 10is prepared according to the procedure described in Example 1. Thereaction medium 83 has six reaction zones 20 ₁–20 ₆, which containpolystryene latexes coated respectively with cocaine (20 ₁), opiates (20₂), PCP (20 ₃), methamphetamine (20 ₄), tetrahyddrocannabinol (20 ₅) andalcohol (20 ₆) as the analytes and a central control zone 16 containingpolystyrene particles, coated with goat anti-mouse immunoglobulin G,embedded in the membrane (as shown in FIG. 1). A sterile swab 85 isplaced under the tongue of a person whose saliva is to be tested for thepresence or absence of the six analytes. After one minute, the swab 85is removed and is placed in the incubation channel 80 and basin 81 ofthe immunoassay device (as shown in FIG. 9). Three drops of a dilutesolution of silver coated gold microsome conjugates of mouseanti-analyte immunoglobulin G for each of the six different analytes(hereinafter “mouse anti-analyte IgG Solution II”) are added to thesaliva swab. The treated swab is allowed to incubate for one minutebefore a finger 82 of the person providing the saliva sample is pressedagainst the swab for ten seconds. The finger is then lifted off theswab, immediately pressed on the reaction medium of the immunoassaydevice and held in place for thirty seconds. The finger is then liftedoff the reaction medium and the medium is allowed to incubate for twominutes before the result is read. At this, point the central controlzone 16 contains the imprint of the finger of the person providing thesaliva sample and reaction zones 20 ₁–20 ₆ are all colored, showing anegative test result presence of all six analytes.

The test procedure is repeated using a new sterile swab except thatprior to the addition of the mouse anti-analyte IgG Solution II to theswab, a drop of a solution containing minor amounts of methamphetamine,tetrahydrocannabinol and alcohol is added to the swab. After completionof the test, the immunoassay device exhibits the result shown in FIG. 2.Central control zone 16 contains the imprint of the finger of the personproviding the saliva sample. Reaction zones 20 ₁–20 ₃ are colored(negative result) and reaction zones 20 ₄–20 ₆ are white (positiveresult).

Example 6

An immunoassay device is prepared as in Example 5. A saliva sample iscollected from under the tongue of a person to be tested for thepresence or absence of the six analytes using a sterile swab. While thesaliva sample is being collected, 800 microliters of mouse anti-analyteIgG Solution I is placed in a sample tube together with one drop of adilute solution of PCP. The saliva-containing swab is allowed to soak inthe solution in the sample tube for two minutes. A 400 microliteraliquot of the resulting mixture is applied to the reaction medium andallowed to incubate for twenty seconds. A 400 microliter aliquot ofSolution A was then added to the reaction medium and allowed to drain.After the Solution A had finished draining, a finger of the person whohad provided the saliva sample is painted with 15 microliters of GoldLabel Solution. The finger is immediately pressed gently against thereaction medium of the immunoassay device and held in place for fiveseconds. After the finger is carefully rolled off the reaction medium,the medium is allowed to incubate for fifteen seconds before another 400microliter aliquot of Solution A is added to the reaction medium. Afterthe completion of the procedure, the reaction zone is completely white,showing a positive test result for PCP. The central control zonecontains an imprint of the finger of the person who had provided thesaliva sample.

Example 7

An immunoassay device is prepared according to the procedure ofExample 1. The reaction medium comprises a nylon membrane. The reactionzone includes polystyrene particles coated with human serumalbumin-cocaine embedded in the membrane. The central control zoneincludes polystyrene particles, coated with goat anti-mouseimmunoglobulin G, embedded in the membrane.

A finger of a person to be tested for the presence or absence of cocainein their blood is pricked with a needle to obtain a droplet of blood.The droplet is spread with a sterile spatula to form a thin film ofblood over the fingertip. The blood covered fingertip is pressed againstthe reaction medium and held in place for about ten seconds. After thefinger is lifted off, the reaction medium is treated with a dilutesolution of household detergent, thereby removing the red blood colorfrom the reaction medium. After the red blood color has been removed,the reaction medium is treated with a dilute solution of alkalinephosphatase conjugated mouse anti-cocaine immunoglobulin G and allowedto incubate for two minutes. The reaction medium is then treatedsequentially with Solution A, a dilute solution of5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium(BCIP/NBT) and again with Solution A. The reaction medium is allowed toincubate for several minutes between the BCIP/NBT treatment and thefinal Solution A treatment. When the procedure is completed, the centralcontrol zone contained an imprint of the finger of the person who hadprovided the blood sample and the reaction zone was colored, showing anegative test result for cocaine.

Example 8

An immunoassay device is prepared as in Example 7. A 500 microliteraliquot of a dilute solution of mouse anti-cocaine immunoglobulin G in aphosphate buffered saline containing 0.1% bovine serum albumin (BSA) and0.05% TWEEN-20 (hereinafter “mouse anti-cocaine IgG Solution”), isplaced in a small sample tube. One drop of a solution containing aminoramount of cocaine is added to the sample tube.

A finger of a person to be tested for the presence or absence of cocainein their blood is pricked with a needle to obtain a droplet of blood andtwo droplets of blood are added to the mixture in the sample tube. Afterthe resulting mixture is allowed to incubate for two minutes in thesample tube, the mixture is added to the reaction medium of theimmunoassay device. The mixture is incubated for one minute on thereaction medium, which is then rinsed with Solution A. A finger of theperson who had provided the blood sample is painted with 15 microlitersof Gold Label Solution. The finger is gently pressed against thereaction medium of the immunoassay device, held in place for fiveseconds and then carefully rolled off the reaction medium. The reactionmedium is allowed to incubate for fifteen seconds before another 400microliter aliquot of Solution A is added to the reaction medium. Aftercompletion of the procedure, the reaction zone is completely white(positive test for cocaine) and the control zone contains the imprint ofthe finger of the person providing the blood sample.

Example 9

An immunoassay device is prepared according to the procedure of Example1 except that the device contains six reaction zones (see FIG. 1). Eachreaction zone contains polystyrene particles coated with a differentamount of cholesterol conjugate embedded in the membrane. Reaction zone20 ₁ contains a cholesterol conjugate which will produce a positivesignal when contacted with a biological fluid having greater than orequal to 150 milligram/deciliter total cholesterol level. Reaction zones20 ₂–20 ₅ contain intermediate amounts of cholesterol conjugate, whereinthe amount of cholesterol conjugate increases sequentially from zone 20₁ to zone 20 ₄, i.e., 180 mg/dl, 240 mg/dl, 280 mg/dl. The reactionmedium also includes two reference control zones corresponding to 30ml/dl and 65 ml/dl high density lipoprotein.

A finger of a person to be tested for the amount of cholesterol in theirblood is pricked with a needle to obtain a droplet of blood. The dropletis added to a small sample tube containing 400 microliters of a dilutesolution of mouse anti-cholesterol immunoglobulin G and colloidal goldconjugated mouse anti-cholesterol immunoglobulin G in a phosphatebuffered saline containing 0.1% bovine serum albumin (BSA) and 0.05%TWEEN-20 (hereinafter “mouse anti-cholesterol IgG Solution”). Afterincubation for three minutes, a thin film of the mixture is spread overa fingertip of the person providing the blood sample using a sterilespatula. The fingertip is immediately gently pressed against thereaction medium of the immunoassay device. After holding the finger inplace for five seconds, the finger is carefully lifted off the reactionmedium. The reaction medium is incubated for one minute and then treatedwith a dilute solution of household detergent to remove red blood colorfrom the reaction medium. After completion of the procedure, the controlzone contains the imprint of the finger of the person providing theblood sample. Reaction zones 20 ₁–20 ₂ are colored (negative test forcholesterol), while reaction zones 20 ₃ and 20 ₄ are white (positivetest for cholesterol). Each reaction zone gives a positive test forcholesterol when at least the predetermined threshold amount ofcholesterol is present. The threshold cholesterol concentration differsfor each reaction zone in correspondence to the amount of cholesterolconjugate adsorbed in the reaction medium in that zone.

Example 10

A listing of a number of other drugs, the presence or absence of whichmay be determined using the present invention follows. This is onlyexemplary and is not meant to limit the invention thereto.

β-Blockers Acebutolol Alprenolol Atenolol Labetalol Metoprolol NadololOxprenolol Propanolol Sotalol and related compounds StimulantsAmfepramone Amphetamine Amphetaminil Amiphenazole Benzphetamine CaffeineCathine Chlorphentermine Clobenzorex Clorprenaline Cocaine CropropamideCrotethamide Dimethamphetamine Ephedrine Etafedrine EthamivanEtilamphetamine Fencamfamin Fenethylline Fenproporex FurfenorexMefenorex Methamphetamine Methoxyphenamine MethylephedrineMethylphenidate Morazone Nikethamide Pemoline PentetrazolPhendimetrazine Phenmetrazine Phentermine Phenylpropanolamine PipradrolProlintane Propylhexedrine Pyrovalerone strychnine and related compoundsAnabolic steroids Bolasterone Boldenone ClostebolDehydromethyltestosterone Fluoxymesterone Mesterolone MethandienoneMethandrostenolone Methenolone Methyltestosterone NandroloneNorethandrolone Oxandrolone Oxymesterone Oxymetholone StanozololTestosterone and related compounds Opiates Heroin Morphine MethandoneMeperidine Codeine Propoxyphene Barbiturates Amobarbital PentobarbitalSecobarbital Phenobarbital Butalbital Butabartial Antipsychotics/Antidepressants Chlorpromazinc Trazodone Haloperidol Amoxapine LithiumCarbonate Imipramine Analgesics Acetylsalicylic Acid AcetaminophenIbuprofen Diflunisal Phenylbutazone Diuretics Acetazolamide AmilondeBendroflumethiazide Bumetanide Canrenone Chlormerodrin ChlorthalidoneDiclofenamide Ethacrynic acid Furosemide Hydrochlorothiazide MersalylSpironolactone Triamterene and related compounds Narcotic analgesicsAlphaprodine Anileridine Suprenorphine Codeine DextromoramideDextropropoxyphene Diamorphine Dihydrocodeine Dipipanone EthoheptazineEthylmorphine Levorphanol Methadone Morphine Nalbuphine PentazocinePethidine Phenazocine Trimeperidine and related compounds HallucinogensLysergic Acid Diethylamide Mescaline Phencyclidine (PCP) Ketamine2,5-Dimethoxy-4-Methylamphetamine Tetrahydrocannabinol MarijuanaSedatives/Hypnotics Chloral Hydrate Glutethimide MeprobamateMethaqualone Benzodiazepines Diazepam Clorazepate ChlordiazepoxideOxazepam Flurazepam Lorazepam Alprazolam Solvents Ethanol MethanolIsopropanol Ethylene Glycol Chloroform Anabolic Steroids TestosteroneMethyltesiosterone Nandrolone Stanozolol Oxandrolone MethandrostenoloneClostebol Mesterolone Norethandrolone

Example 11

The following table sets forth exemplary chromogenic substrates yieldingwater-insoluble products that may be used with an appropriate enzymeconjugate in the invention, instead of the colloidal gold labelpreviously noted. Labeling methods utilizing enzyme/chromogen couplesare well known and would be easily practiced by one skilled in the art.

Chromogenic Substrates Yielding Water-Insoluble Products

Chromogenic Substrates Yielding Water-Insoluble Products Orig. FinalEnzyme Substrate Abbreviation Color Color Horseradish DiaminobenzideneDAB Clear Brown Peroxidase Diaminobenzidene DAB/nickel Clear Grey/ withnickel Black enhancement 3-Amino-9- AEC Clear Red ethylcarbazole4-Chloro-1-naphthol — Clear Blue Alkaline Naphthol-AS-BI- NABP/FR ClearRed Phosphatase phosphate/fast red TR Naphthol-AS-MX- NAMP/FR Clear Redphosphate/fast red TR Naphthol-AS-BI- NABP/NF Clear Red phosphate/newfuchsin Bromochloroindolyl BCIP/NBT Purple Clear phosphate/nitro-bluetetrazolium 5-Bromo-4-chloro-3- BCIG Clear Blue indolyl-B-d-galactopyranoside B- Naphthol AS-BI-B-d- NABG Clear Red Galactosidasegalactopyranoside

Example 12

In this example, the device and method of Example 7 were used, exceptthat the assay device included reaction zones for the analytes, shown inExample 3, the surface of the reaction medium was pre-wetted withSolution A, the test subject's finger was pricked to produce asufficient quantity of blood to smear the blood over the tip of thesubject's finger, and the smeared blood was allowed to dry on thesubject's finger. The finger with the dried blood was then applied tothe surface of assay device and held in contact with the device forabout 15 seconds. After the finger is lifted off, the reaction mediumwas treated with a dilute solution of clearing reagent, thereby removingthe red blood color from the reaction medium. After the red blood colorwas removed, the reaction medium is treated with a dilute solution ofconjugates of colloidal gold bonded to a secondary antibody, eachconjugate specifically binding to one of the named analytes. Afterincubation for about two minutes, a fingertip which had not been used toapply the blood was coated with about 10 microliters of colloidal goldconjugated to a ligand specific for a ligand receptor in the controlzone. After the fingertip was removed, the central control zonecontained an imprint of the finger of the person who had provided theblood sample and the reaction zone was colored, showing a negative testresult for the analytes.

Example 13

In this example, the device and method of Example 7 were used, exceptthat the assay device included reaction zones for the analytes, shown inExample 3, the surface of the reaction medium was dry, the testsubject's finger was pricked to produce a sufficient quantity of bloodto smear the blood over the tip of the subject's finger, and the smearedblood was immediately (before drying) applied to the reaction medium forabout 15 seconds. After the finger is lifted off, the reaction mediumwas allowed to dry (typically about 1 to 2 minutes), treated with aclearing solution to remove the red blood cells, and treated with adilute solution of clearing reagent, thereby removing the red bloodcolor from the reaction medium. After the red blood color was removed,the reaction medium is treated with a dilute solution of conjugates ofcolloidal gold bonded to a secondary antibody, each conjugatespecifically binding to one of the named analytes. After incubation forabout two minutes, a fingertip which had not been used to apply theblood was coated with about 10 microliters of colloidal gold conjugatedto a ligand specific for a ligand receptor in the control zone. Afterthe fingertip was removed, the central control zone contained an imprintof the finger of the person who had provided the blood sample and thereaction zone was colored, showing a negative test result for theanalytes.

Example 14

An assay device, containing five reaction zones (corresponding to areas20 ₁ and 20 ₃–20 ₆ in FIG. 1), is prepared according to the procedure ofExample 1. As in Example 3, the reaction zones contain polystyrenelatexes coated respectively with cocaine (20 ₁), PCP (20 ₃),amphetamine/methamphetamine (20 ₄), tetrahydrocannabinol (20 ₅) andalcohol (20 ₆) as the analytes. In this instance, however, zone 20 ₂does not contain any adsorbed analyte or antibody and serves as areference control zone, i.e., a zone which will remain completely whiteat the conclusion of the test. The central control zone (“identitycontrol zone” corresponding to zone 16 in FIG. 1) is prepared as inExample 1, except that it contains polystyrene particles coated withgoat anti-rabbit immunoglobulin G embedded in the membrane.

The reaction medium is treated with 500 microliters of Solution A,thereby wetting the reaction medium. A sample of blood is obtained froma test subject to test for the presence or absence of the five analytesin their blood. A portion of the sample is coated on the test subject'sfingertip and applied to the five reaction zones and the referencecontrol zone. Pressure is maintained on the reaction medium for about10–15 seconds.

A few drops, e.g., about a 500 microliter aliquot of a dilute householddetergent solution is then added to the reaction medium and allowed todrain. The household detergent treatment lyses any red blood cellspresent in the reaction medium and rinses the dark red color from theblood out of the medium. The five reaction zones and the referencecontrol zone are then treated with about 10 ml of a dilute solution of amixture of colloidal gold conjugated mouse anti-analyte immunoglobulin Gin a TRIS buffer containing 1.0% BSA, 0.05% TWEEN-20 and 0.05% azide.

A finger of the person who had provided the blood sample is then paintedwith 15 microliters of a dilute solution of colloidal gold conjugatedrabbit anti-goat immunoglobulin G in a TRIS buffer containing 1.0% BSA,0.05% TWEEN-20 and 0.05% azide. The finger is gently pressed against theidentity control zone of the reaction medium and held in place for threeseconds. After the finger is carefully lifted off the reaction medium,the medium is allowed to incubate for fifteen seconds before another 400microliter aliquot of Solution A is added to the reaction medium. Aftercompletion of the procedure, the reaction zones 20 ₁ and 20 ₃–20 ₆ arecolored, showing a negative test result for the analytes. The referencecontrol zone 20 ₂ is completely white and the identity control zonecontains the imprint of the finger of the person providing the bloodsample.

Although the present invention has been described in terms of exemplaryembodiment, it is not limited to these embodiments. Alternativeembodiments, examples, and modifications which would still beencompassed by the invention may be made by those skilled in the art,particularly in light of the foregoing teachings. Therefore, thefollowing claims are intended to cover any alternative embodiments,examples, modifications, or equivalents which may be included within thespirit and scope of the invention as defined by the claims.

1. A method for detecting an analyte in a test sample provided by a testsubject having an identifying body part surface and simultaneouslyestablishing the identity of said test subject comprising: (a) providinga self-contained assay device having an identifying control zone, a testsample medium, a reaction medium containing a member of an immunologicalpair, and a signal-producing agent, wherein said test sample medium andsaid reaction medium are moveable between a first position in which thetest sample medium and the reaction medium are in a spaced relationshipand a second position in which the test sample medium and reactionmedium are in sample-transferring contact and wherein said signalproducing agent and said reaction medium are movable between a firstposition in which the signal producing agent and the reaction medium arein a spaced relationship and a second position in which the signalproducing agent and the reaction medium are in signal producingagent-transferring contact and wherein both said test sample medium andsaid signal producing agent are in a spaced relationship to saidreaction medium; (b) contacting said test sample with said test samplemedium, wherein at least a portion of said test sample is transferred tosaid test sample medium; (c) moving said test sample medium to saidsecond test sample-transferring contact position with said reactionmedium, wherein at least a portion of said test sample is transferredfrom said test sample medium to said reaction medium and wherein anyanalyte present in said test sample forms a conjugate with the member ofan immunological pair in said reaction medium; (d) moving said testsample medium to said first spaced relationship to said reaction medium;(e) moving said signal-producing agent to said second signal-producingagent-transferring contact position with said reaction medium, whereinat least a portion of said signal producing agent is transferred to saidreaction medium and wherein said signal-producing agent binds to saidreaction medium in the presence of conjugated analyte; and (f)determining the presence or absence of said analyte by detecting thepresence or absence of bound signal-producing agent; said method furthercomprising (g) contacting said signal-producing agent with saididentifying body part surface of said test subject wherein at least aportion of said signal-producing agent is transferred to saididentifying body part surface; and (h) placing said identifying bodypart surface in signal-producing agent-transferring contact with saididentifying control zone wherein at least a portion of saidsignal-producing agent is transferred to said identifying control zonesuch that an identifying imprint of said identifying body part surfaceis created on said identifying control zone to establish the identity ofsaid test subject.
 2. The method according to claim 1, furthercomprising the step of treating said test sample medium with dilutedetergent after step (c).
 3. A method for detecting an analyte in a testsample provided by a test subject having an identifying body partsurface and simultaneously establishing the identity of said testsubject comprising: (a) providing a self-contained assay device having asignal-producing agent and a reaction medium containing a member of animmunological pair, wherein said signal producing agent and saidreaction medium are movable between a first position in which the signalproducing agent and the reaction medium are in a spaced relationship anda second position in which the signal producing agent and the reactionmedium are in signal producing agent-transferring contact and whereinsaid signal producing agent is in a spaced relationship to said reactionmedium, and an identifying control zone; (b) contacting said test samplewith said identifying body part surface of said test subject, wherein atleast a portion of said test sample is transferred to said identifyingbody part surface; (c) placing said identifying body part surface intest sample-transferring contact with said reaction medium, wherein atleast a portion of said test sample is transferred from said identifyingbody part surface to said reaction medium, wherein any analyte presentin said test sample forms a conjugate with the member of animmunological pair in said reaction medium; (d) removing saididentifying body part surface to a position in spaced relationship tosaid reaction medium; (e) moving said signal-producing agent to saidsecond signal producing agent-transferring contact position with saidreaction zone, wherein at least a portion of said signal producing agentis transferred to said reaction medium and wherein said signal-producingagent binds to said reaction medium in either the presence or absence ofconjugated analyte; and (f) determining the presence or absence of saidanalyte by detecting the presence or absence of bound signal-producingagent; said method further comprising (g) contacting saidsignal-producing agent with said identifying body part surface of saidtest subject wherein at least a portion of said signal-producing agentis transferred to said identifying body part surface; and (h) placingsaid identifying body part surface in signal-producingagent-transferring contact with said identifying control zone wherein atleast a portion of said signal-producing agent is transferred to saididentifying control zone such that an identifying imprint of saididentifying body part surface is created on said identifying controlzone to establish the identity of said test subject.
 4. The methodaccording to claim 3, further comprising the step of treating said testsample medium with dilute detergent after step (d).
 5. A method fordetecting an analyte in a test sample provided by a test subject havingan identifying body part surface and simultaneously establishing theidentity of said test subject comprising: (a) providing a self-containedassay device having an identifying control zone, a test sample medium, areaction medium containing a member of an immunological pair, and asignal-producing agent, wherein said test sample medium and saidreaction medium are moveable between a first position in which the testsample medium and the reaction medium are in a spaced relationship and asecond position in which the test sample medium and reaction medium arein sample-transferring contact and wherein said signal producing agentand said reaction medium are movable between a first position in whichthe signal producing agent and the reaction medium are in a spacedrelationship and a second position in which the signal producing agentand the reaction medium are in signal producing agent-transferringcontact and wherein both said test sample medium and said signalproducing agent are in a spaced relationship to said reaction medium;(b) contacting said test sample with said test sample medium, wherein atleast a portion of said test sample is transferred to said test samplemedium; (c) moving said test sample medium to said second testsample-transferring contact position with said reaction medium, whereinat least a portion of said test sample is transferred from said testsample medium to said reaction medium and wherein any analyte present insaid test sample forms a conjugate with the member of an immunologicalpair in said reaction medium; (d) moving said test sample medium to saidfirst spaced relationship to said reaction medium; (e) moving saidsignal-producing agent to said second signal-producingagent-transferring contact position with said reaction medium, whereinat least a portion of said signal producing agent is transferred to saidreaction medium and wherein said signal-producing agent binds to saidreaction medium in the absence of conjugated analyte; and (f)determining the presence or absence of said analyte by detecting thepresence or absence of bound signal-producing agent; said method furthercomprising (g) contacting said signal-producing agent with saididentifying body part surface of said test subject wherein at least aportion of said signal-producing agent is transferred to saididentifying body part surface; and (h) placing said identifying bodypart surface in signal-producing agent-transferring contact with saididentifying control zone wherein at least a portion of saidsignal-producing agent is transferred to said identifying control zonesuch that an identifying imprint of said identifying body part surfaceis created on said identifying control zone to establish the identity ofsaid test subject.
 6. A method for detecting an analyte in a test sampleprovided by a test subject having an identifying body part surface andsimultaneously establishing the identity of said test subjectcomprising: (a) providing a self-contained assay device having asignal-producing agent and a reaction medium containing a member of animmunological pair, wherein said signal producing agent and saidreaction medium are movable between a first position in which the signalproducing agent and the reaction medium are in a spaced relationship anda second position in which the signal producing agent and the reactionmedium are in signal producing agent-transferring contact and whereinsaid signal producing agent is in a spaced relationship to said reactionmedium, and an identifying control zone; (b) contacting said test samplewith said identifying body part surface of said test subject, wherein atleast a portion of said test sample is transferred to said identifyingbody part surface; (c) placing said identifying body part surface intest sample-transferring contact with said reaction medium, wherein atleast a portion of said test sample is transferred from said identifyingbody part surface to said reaction medium, wherein any analyte presentin said test sample forms a conjugate with the member of animmunological pair in said reaction medium; (d) removing saididentifying body part surface to a position in spaced relationship tosaid reaction medium; (e) moving said signal-producing agent to saidsecond signal producing agent-transferring contact position with saidreaction zone, wherein at least a portion of said signal producing agentis transferred to said reaction medium and wherein said signal-producingagent binds to said reaction medium in the absence of conjugatedanalyte; and (f) determining the presence or absence of said analyte bydetecting the presence or absence of bound signal-producing agent; saidmethod further comprising (g) contacting said signal-producing agentwith said identifying body part surface of said test subject wherein atleast a portion of said signal-producing agent is transferred to saididentifying body part surface; and (h) placing said identifying bodypart surface in signal-producing agent-transferring contact with saididentifying control zone wherein at least a portion of saidsignal-producing agent is transferred to said identifying control zonesuch that an identifying imprint of said identifying body part surfaceis created on said identifying control zone to establish the identity ofsaid test subject.